The overall objective of this proposal is to characterize the immune response against a new HIV vaccine design composed of a mixture of biodegradable nanospheres to deliver the HIV antigen proteins as well as the plasmids that encode these proteins, and biodegradable microspheres to provide a local sustained delivery of cytokines. DNA immunization of HIV-1 gene products have induced potent humoral and cell-mediated immune responses in rodents and non-human primates. The plasmids are typically administered as bolus injections or through a gene gun. The applicants have developed a non-viral gene delivery system based on DNA nanospheres synthesized by salt-induced complex coacervation of DNA with either gelatin or chitosan. This gene delivery system has several useful features, namely: (1) ligands can be conjugated to the nanosphere to stimulate receptor-mediated endocytosis; (2) other bioactive agents or multiple plasmids can be co-encapsulated; (3) bioavailability of the DNA can be improved; and (4) the nanosphere can be lyophilized for storage without loss of bioactivity. It has been reported that antigens conjugated to beads, such as polystyrene particles can be targeted into the phagocytic pathway to induce protective tumor immunity. Particulate antigens, compared to soluble antigens, have also been shown to be more effectively presented by lymphocytes for MHC class II presentation. In the proposed HIV vaccine design, the recombinant HIV-1 antigen proteins can be co-encapsulated in the DNA nanospheres described above, with the potential of augmenting the immune response elicited by the DNA inoculation. To enhance and to steer the type of immune response (Thl versus Th2), the researchers have codelivered cytokines with HIV vaccine constructs. However, it may be difficult to control the cytokine delivery achieved by transient non-viral transfection. If administered by bolus injection, previous studies on tumor vaccines have shown that the cytokines would be cleared from the site of injection within hours. A microsphere-controlled release formulation can maintain a high level of cytokines local to the vaccine site for days, providing the co-stimulating signals for the infiltrating lymphocytes. The hypothesis of this proposal is that application of the controlled release technology to deliver the HIV-1 genes, recombinant HIV-1 antigen proteins, and cytokines by nanospheres and microspheres will be an effective HIV vaccine design. The specific aims of the proposed work are to synthesize these particulate HIV vaccines and to evaluate their immune response in terms of T cell lymphoproliferation, cytotoxic T cell response, antibody titers, and Thl/Th2 profiles.